ABSTRACT - PROJECT I Successful human pregnancy is believed to rely upon production of HLA-G proteins by trophoblast cells in placentas. The HLA-G gene generates multiple transcripts that encode four membrane and three soluble proteins. Two of the soluble isoforms, HLA-G5 (G5) and HLA-G6 (G6) (also known as sHLA-G1 and SHLA-G2, respectively), are not only present at the maternal-fetal interface but also circulate in maternal blood throughout pregnancy. These proteins appear to be biologically important: the scientific literature documents associations between pregnancy success and high levels of soluble HLA-G in the supernatant culture media of in vitro cultured embryos, and critical preliminary experiments in our laboratory indicate that women who suffer recurrent spontaneous abortions not only fail to increase their serum levels of HLA-G5 but may decrease their levels of HLA-G6 with pregnancy . In order to study the impact of these proteins on the mother's immune responses to her genetically different embryo/fetus, we developed eukaryotic expression vectors and generated monoclonal antibodies specific for the proteins. Studies using these unique reagents have demonstrated that G5 and G6 differ in primary and secondary structures. Expression studies have documented marked differences between the isoforms in their localization to specific subpopulations of trophoblast cells, experiments on regulation of expression have demonstrated both general and isoform-specific regulatory conditions, and studies on function have identified qualitative and quantitative differences between the two isoforms. In this application we propose studies that will expand our knowledge of these powerful, pregnancy-associated proteins. AIM 1 is designed to investigate potential ligand:receptor interactions in mononuclear phagocytes, mapping expression, and performing binding studies. The goal of AIM 2 is to establish mechanisms of regulation of differential expression of G5 and G6 proteins in subpopulations of trophoblast cells from early and late gestation placentas. In AIM 3 the purpose is to investigate how G5 and G6 function to program mononuclear phagocytes. We will systematically compare results on samples from 1st trimester with term placentas and results on normal placentas with age-matched samples from problem pregnancies, i.e., preeclampsia and preterm labor/delivery with and without infection. The relevance of the proposed research to public health rests on the fact that at least one in ten couples experiences fertility difficulties and a high proportion of pregnancies fail due to preterm labor associated or not with infection. One underlying cause may be aberrancies of synthesis or expression of G5/G6 or their LILRB1/LILRB2 receptors. We expect here to provide entirely novel information that is essential to the design of therapeutic strategies to address HLA-G-associated pathologies of pregnancy.